RESEARCH ARTICLE
Müller Glia Activation in Response to Inherited Retinal Degeneration Is Highly Varied and Disease-Specific Claire Hippert1☯, Anna B. Graca1☯, Amanda C. Barber1¤, Emma L. West1, Alexander J. Smith1, Robin R. Ali1,2, Rachael A. Pearson1*
a11111
1 Department of Genetics, University College London Institute of Ophthalmology, 11–43 Bath Street, London, EC1V 9EL, United Kingdom, 2 NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, City Road, London, EC1V 2PD, United Kingdom ☯ These authors contributed equally to this work. ¤ Current address: John van Geest Centre for Brain Repair, University of Cambridge, ED Adrian Building, Forvie Site, Robinson Way, Cambridge, CB2 0PY, United Kingdom * [email protected]
OPEN ACCESS Citation: Hippert C, Graca AB, Barber AC, West EL, Smith AJ, Ali RR, et al. (2015) Müller Glia Activation in Response to Inherited Retinal Degeneration Is Highly Varied and Disease-Specific. PLoS ONE 10(3): e0120415. doi:10.1371/journal.pone.0120415 Academic Editor: Rafael Linden, Universidade Federal do Rio de Janeiro, BRAZIL Received: November 14, 2014 Accepted: January 22, 2015 Published: March 20, 2015 Copyright: © 2015 Hippert et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by grants from Fight For Sight (http://www.fightforsight.org.uk/ grant number: 1448/1449), the Medical Research Council UK (http://www.mrc.ac.uk/ grant number: mr/j004553/ 1), and Retinitis Pigmentosa Fighting Blindness (http://www.rpfightingblindness.org.uk/ grant number: GR566). A.B.G. is an MRC-DTA Clinical Neuroscience Ph.D. student. R.R.A. is part-funded by National Institute for Health Research (NIHR) Biomedical Research Centre for Ophthalmology at Moorfields Eye Hospital (http://www.moorfields.nhs.
Abstract Despite different aetiologies, most inherited retinal disorders culminate in photoreceptor loss, which induces concomitant changes in the neural retina, one of the most striking being reactive gliosis by Müller cells. It is typically assumed that photoreceptor loss leads to an upregulation of glial fibrilliary acidic protein (Gfap) and other intermediate filament proteins, together with other gliosis-related changes, including loss of integrity of the outer limiting membrane (OLM) and deposition of proteoglycans. However, this is based on a mix of both injury-induced and genetic causes of photoreceptor loss. There are very few longitudinal studies of gliosis in the retina and none comparing these changes across models over time. Here, we present a comprehensive spatiotemporal assessment of features of gliosis in the degenerating murine retina that involves Müller glia. Specifically, we assessed Gfap, vimentin and chondroitin sulphate proteoglycan (CSPG) levels and outer limiting membrane (OLM) integrity over time in four murine models of inherited photoreceptor degeneration that encompass a range of disease severities (Crb1rd8/rd8, Prph2+/Δ307, Rho-/-, Pde6brd1/rd1). These features underwent very different changes, depending upon the disease-causing mutation, and that these changes are not correlated with disease severity. Intermediate filament expression did indeed increase with disease progression in Crb1rd8/rd8 and Prph2+/Δ307, but decreased in the Prph2+/Δ307 and Pde6brd1/rd1 models. CSPG deposition usually, but not always, followed the trends in intermediate filament expression. The OLM adherens junctions underwent significant remodelling in all models, but with differences in the composition of the resulting junctions; in Rho-/- mice, the adherens junctions maintained the typical rod-Müller glia interactions, while in the Pde6brd1/rd1 model they formed predominantly between Müller cells in late stage of degeneration. Together, these results show that gliosis and its associated processes are variable and disease-dependent.
PLOS ONE | DOI:10.1371/journal.pone.0120415 March 20, 2015
1 / 27
Gliosis in Inherited Retinal Degeneration
uk/), UCL Institute of Ophthalmology (http://www.ucl. ac.uk/ioo/), and the Alcon Research Institute (http:// www.alcon.com/en/ARI/index.asp). R.A.P. is a Royal Society University Research Fellow (https:// royalsociety.org/) and supported by the Alcon Research Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: R.A.P. and R.R.A. are each in receipt of an Alcon Research Institute award. These are unrestricted research grants. There are no patents, products in development, or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
Introduction The vast majority of degenerative retinal diseases lead either directly or indirectly to the loss of photoreceptor cells. As degeneration progresses so the microenvironment of the retina undergoes a number of significant changes. The loss of photoreceptors causes the cytoarchitecture of the outer nuclear layer (ONL) to become disrupted and the outer limiting membrane (OLM), a network of adherens junctions formed between photoreceptors and Müller glia, may become compromised. In addition, Müller glial cells undergo reactive gliosis, leading to the formation of a glial scar that can envelope the entire retina at late stages of degeneration [1,2]. This scar can act as a reservoir for the accumulation of extracellular matrix (ECM) proteins including chondroitin sulphate proteoglycans (CSPGs), which are known to be inhibitory to axonal regeneration [3,4]. Each of these processes is likely to have a significant impact upon the retina and its health and physiology. Moreover, having a complete understanding of such changes is essential for the development of promising new therapeutic strategies including gene [5] and cell [6] replacement. Previously, retinal gliosis has been shown to negatively impact on the efficiency of viral transduction in gene therapy [7], the integration of transplanted photoreceptors ([8], reviewed in [9]) and the ability of retinal grafts [10] and electronic implants [11] to contact the underlying retina. Elsewhere in the CNS, reactive gliosis has long been considered as the major impediment to axonal regrowth after an injury [12,13]. Nonetheless, the formation of a glial barrier around a lesion site is also an advantage, because it isolates the still intact CNS tissue from secondary lesions. In addition, there are reports to suggest that in certain conditions reactive astrocytes could even provide a permissive substratum for neurite extension (reviewed in [14]). For these reasons, understanding the process of glial scar formation, how this process differs in different models of degeneration, and finding strategies to circumvent these barriers, represent major challenges to the advancement of many ocular therapies. Typically, gliosis is characterized by a dramatic increase in intermediate filament expression and a pronounced hypertrophy of Müller cells [15]. In addition to the upregulation of the intermediate filament proteins, glial fibrillary acidic protein (Gfap) and vimentin, reactive Müller cells may undergo hypertrophy, presenting a proliferation of fibrous processes and deposition of proteoglycans, particularly CSPGs, at the outer edge of the retina [16–18]. This process of gliosis is characteristic of many retinal disease models [19–21], although the temporal relationship between the onset of gliosis and degeneration may vary between disease models. To our knowledge, no comparison between models at equivalent stages of degeneration has been made. Given the apparent complexities of the gliotic process, more precise dissections of the links between the onset of glial reactivity and progressive neurodegeneration are needed. Here, we present the first comparative characterization of the changes in the microenvironment of the neural retina in four models of inherited retinal degeneration over time. Specifically, we assess the levels of Gfap, vimentin and CSPGs, together with OLM integrity and ONL architecture, to provide a more comprehensive analysis of the presence and distribution of these proteins in the degenerating retina. Our data show that different initiating genetic defects can lead to striking differences in the response by Müller glia.
Materials and Methods Ethics statement All animal studies were carried out under the Animals (Scientific Procedures) Act 1986 under a project license PPL 70/8120 issued by the UK Government Home Office and conducted in accordance with protocols approved by the Animal Welfare and Ethics Committee of the UCL Institute of Ophthalmology. All animals were killed by trained personnel using cervical
PLOS ONE | DOI:10.1371/journal.pone.0120415 March 20, 2015
2 / 27
Gliosis in Inherited Retinal Degeneration
Table 1. Summary of the different models and stages of retinal degeneration studied. Mouse model
Early Degeneration (ONL >70% of WT)
Mid Degeneration (ONL 30–70% of WT)
Late Degeneration (ONL 70% of the thickness of the ONL in wild-type retinae, mid-degeneration, where ONL thickness is 30–70% of wild-type and late degeneration, where it is reduced to
Müller Glia Activation in Response to Inherited Retinal Degeneration Is Highly Varied and Disease-Specific Claire Hippert1☯, Anna B. Graca1☯, Amanda C. Barber1¤, Emma L. West1, Alexander J. Smith1, Robin R. Ali1,2, Rachael A. Pearson1*
a11111
1 Department of Genetics, University College London Institute of Ophthalmology, 11–43 Bath Street, London, EC1V 9EL, United Kingdom, 2 NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, City Road, London, EC1V 2PD, United Kingdom ☯ These authors contributed equally to this work. ¤ Current address: John van Geest Centre for Brain Repair, University of Cambridge, ED Adrian Building, Forvie Site, Robinson Way, Cambridge, CB2 0PY, United Kingdom * [email protected]
OPEN ACCESS Citation: Hippert C, Graca AB, Barber AC, West EL, Smith AJ, Ali RR, et al. (2015) Müller Glia Activation in Response to Inherited Retinal Degeneration Is Highly Varied and Disease-Specific. PLoS ONE 10(3): e0120415. doi:10.1371/journal.pone.0120415 Academic Editor: Rafael Linden, Universidade Federal do Rio de Janeiro, BRAZIL Received: November 14, 2014 Accepted: January 22, 2015 Published: March 20, 2015 Copyright: © 2015 Hippert et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by grants from Fight For Sight (http://www.fightforsight.org.uk/ grant number: 1448/1449), the Medical Research Council UK (http://www.mrc.ac.uk/ grant number: mr/j004553/ 1), and Retinitis Pigmentosa Fighting Blindness (http://www.rpfightingblindness.org.uk/ grant number: GR566). A.B.G. is an MRC-DTA Clinical Neuroscience Ph.D. student. R.R.A. is part-funded by National Institute for Health Research (NIHR) Biomedical Research Centre for Ophthalmology at Moorfields Eye Hospital (http://www.moorfields.nhs.
Abstract Despite different aetiologies, most inherited retinal disorders culminate in photoreceptor loss, which induces concomitant changes in the neural retina, one of the most striking being reactive gliosis by Müller cells. It is typically assumed that photoreceptor loss leads to an upregulation of glial fibrilliary acidic protein (Gfap) and other intermediate filament proteins, together with other gliosis-related changes, including loss of integrity of the outer limiting membrane (OLM) and deposition of proteoglycans. However, this is based on a mix of both injury-induced and genetic causes of photoreceptor loss. There are very few longitudinal studies of gliosis in the retina and none comparing these changes across models over time. Here, we present a comprehensive spatiotemporal assessment of features of gliosis in the degenerating murine retina that involves Müller glia. Specifically, we assessed Gfap, vimentin and chondroitin sulphate proteoglycan (CSPG) levels and outer limiting membrane (OLM) integrity over time in four murine models of inherited photoreceptor degeneration that encompass a range of disease severities (Crb1rd8/rd8, Prph2+/Δ307, Rho-/-, Pde6brd1/rd1). These features underwent very different changes, depending upon the disease-causing mutation, and that these changes are not correlated with disease severity. Intermediate filament expression did indeed increase with disease progression in Crb1rd8/rd8 and Prph2+/Δ307, but decreased in the Prph2+/Δ307 and Pde6brd1/rd1 models. CSPG deposition usually, but not always, followed the trends in intermediate filament expression. The OLM adherens junctions underwent significant remodelling in all models, but with differences in the composition of the resulting junctions; in Rho-/- mice, the adherens junctions maintained the typical rod-Müller glia interactions, while in the Pde6brd1/rd1 model they formed predominantly between Müller cells in late stage of degeneration. Together, these results show that gliosis and its associated processes are variable and disease-dependent.
PLOS ONE | DOI:10.1371/journal.pone.0120415 March 20, 2015
1 / 27
Gliosis in Inherited Retinal Degeneration
uk/), UCL Institute of Ophthalmology (http://www.ucl. ac.uk/ioo/), and the Alcon Research Institute (http:// www.alcon.com/en/ARI/index.asp). R.A.P. is a Royal Society University Research Fellow (https:// royalsociety.org/) and supported by the Alcon Research Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: R.A.P. and R.R.A. are each in receipt of an Alcon Research Institute award. These are unrestricted research grants. There are no patents, products in development, or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
Introduction The vast majority of degenerative retinal diseases lead either directly or indirectly to the loss of photoreceptor cells. As degeneration progresses so the microenvironment of the retina undergoes a number of significant changes. The loss of photoreceptors causes the cytoarchitecture of the outer nuclear layer (ONL) to become disrupted and the outer limiting membrane (OLM), a network of adherens junctions formed between photoreceptors and Müller glia, may become compromised. In addition, Müller glial cells undergo reactive gliosis, leading to the formation of a glial scar that can envelope the entire retina at late stages of degeneration [1,2]. This scar can act as a reservoir for the accumulation of extracellular matrix (ECM) proteins including chondroitin sulphate proteoglycans (CSPGs), which are known to be inhibitory to axonal regeneration [3,4]. Each of these processes is likely to have a significant impact upon the retina and its health and physiology. Moreover, having a complete understanding of such changes is essential for the development of promising new therapeutic strategies including gene [5] and cell [6] replacement. Previously, retinal gliosis has been shown to negatively impact on the efficiency of viral transduction in gene therapy [7], the integration of transplanted photoreceptors ([8], reviewed in [9]) and the ability of retinal grafts [10] and electronic implants [11] to contact the underlying retina. Elsewhere in the CNS, reactive gliosis has long been considered as the major impediment to axonal regrowth after an injury [12,13]. Nonetheless, the formation of a glial barrier around a lesion site is also an advantage, because it isolates the still intact CNS tissue from secondary lesions. In addition, there are reports to suggest that in certain conditions reactive astrocytes could even provide a permissive substratum for neurite extension (reviewed in [14]). For these reasons, understanding the process of glial scar formation, how this process differs in different models of degeneration, and finding strategies to circumvent these barriers, represent major challenges to the advancement of many ocular therapies. Typically, gliosis is characterized by a dramatic increase in intermediate filament expression and a pronounced hypertrophy of Müller cells [15]. In addition to the upregulation of the intermediate filament proteins, glial fibrillary acidic protein (Gfap) and vimentin, reactive Müller cells may undergo hypertrophy, presenting a proliferation of fibrous processes and deposition of proteoglycans, particularly CSPGs, at the outer edge of the retina [16–18]. This process of gliosis is characteristic of many retinal disease models [19–21], although the temporal relationship between the onset of gliosis and degeneration may vary between disease models. To our knowledge, no comparison between models at equivalent stages of degeneration has been made. Given the apparent complexities of the gliotic process, more precise dissections of the links between the onset of glial reactivity and progressive neurodegeneration are needed. Here, we present the first comparative characterization of the changes in the microenvironment of the neural retina in four models of inherited retinal degeneration over time. Specifically, we assess the levels of Gfap, vimentin and CSPGs, together with OLM integrity and ONL architecture, to provide a more comprehensive analysis of the presence and distribution of these proteins in the degenerating retina. Our data show that different initiating genetic defects can lead to striking differences in the response by Müller glia.
Materials and Methods Ethics statement All animal studies were carried out under the Animals (Scientific Procedures) Act 1986 under a project license PPL 70/8120 issued by the UK Government Home Office and conducted in accordance with protocols approved by the Animal Welfare and Ethics Committee of the UCL Institute of Ophthalmology. All animals were killed by trained personnel using cervical
PLOS ONE | DOI:10.1371/journal.pone.0120415 March 20, 2015
2 / 27
Gliosis in Inherited Retinal Degeneration
Table 1. Summary of the different models and stages of retinal degeneration studied. Mouse model
Early Degeneration (ONL >70% of WT)
Mid Degeneration (ONL 30–70% of WT)
Late Degeneration (ONL 70% of the thickness of the ONL in wild-type retinae, mid-degeneration, where ONL thickness is 30–70% of wild-type and late degeneration, where it is reduced to
